The present invention relates to compositions and methods for protecting mammalian cells from injury due to intrinsic membrane lysis, oxidation and/or invasion by destructive agents. In particular the present invention relates to compositions and methods for treating against and/or prophylactically inhibiting the injury causation. Even more particularly, the present invention relates to bioactive agents and the use thereof for treating or prophylactically inhibiting phospholipase mediated injury, injury due to oxidation, and inflammation. In a specific sense the present invention provides agents for preventing and/or treating inflammation and cell destruction in mammalian tissue and for protection and preservation of biologic material derived from animals, humans and plants such as food and tissue samples. In a very specific sense, this invention provides compositions that are inhibitors of phospholipase and methods of making these compositions.
The base structure of all living organisms is the cell which is structurally defined by its membranous lipoprotein envelope. The membranous network that holds the cell together maintains the ionic balance and provides the receptors for hormones and neurotransmitters that enable a cell to interact with its environment. This is pertinent to interaction with neighboring cells which enable isolated cells, tissues, or whole organisms to survive as both independent units and as participants in cellular interactions, in vitro and in vivo.
External factors which govern cell function, renewal, reproduction and death act via their effects on the phospholipid bilayer and proteins of the cell membrane. This controls the receptor-mediated signals and ionic fluxes which govern cell responsiveness and survival. Damage to the cell membrane with particular emphasis on lipid peroxidation, membrane oxidation and the action of phospholipases, affects resistance to injury, repair and host responses to environmental change and ionic and osmotic integrity.
Pathological events in a host under clinical circumstances can result in cellular insult, leading to loss of membrane integrity. The events are mediated by factors which digest and destroy cell membrane and propagate an injury by producing a cascade of cell membrane changes. By interfering with the cascade of external and internal events involving membrane integrity and toxic changes which lead to cell death, injury can be prevented, modified or reversed. This has been a major role of anti-inflammatory agents in the past.
The most important presently used clinically effective anti-inflammatory drugs include the corticosteroids and the non-steroidal anti-inflammatory drugs (NSAIDs). Corticosteroids inhibit the activity of cell phospholipases among other actions. NSAIDs inhibit the metabolism by cyclooxygenase of arachidonic acid released by phospholipases. These drugs act to control inflammation and to minimize cell injury by regulating the breakdown of phospholipids. These drugs also affect the action of the products of phospholipid breakdown leading to the formation of prostaglandins and leukotrienes which are produced in increased quantities in inflammation and promote cell dysfunction and injury.
In addition, cellular and extracellular phospholipases may be activated by the generation of oxygen free radicals. This can establish a damaging cycle as phospholipase activation can release free radicals which, in turn, activate more phospholipases. In this regard, free radicals are produced from the fatty acids which are released by the action of phospholipases and then converted to prostaglandins and leukotrienes by cyclooxygenase and lipoxygenase enzymes with oxygen free radical production as a by product. Fatty acids and free radicals are known to be prime mediators in the cascade of reactions that result in membrane injury, cell death and inflammation. Phospholipase A2 (PLA2), a key enzyme in the metabolism of phospholipid, can promote fatty acid release. PLA2 may be activated by a variety of factors involving hormonal, neural, metabolic, or immunologic pathways.
One of the hallmarks of inflammation and cell injury is the breakdown of cellular membrane phospholipid. Phospholipids are the major structural building blocks of the cell membrane; they give rise to the barrier-structural and functional properties of membranes and their integrity is crucial to normal cell responsiveness and function. Phospholipid changes in cell membrane integrity, particularly changes in fatty acids at the 2 position, alter the fluidity of cell membranes, cell receptor function and the availability of cellular contents to the external environment. The breakdown of phospholipid membranes results in lysis of cells, produces holes in the cell membrane, affects ion channels and membrane receptors which destroy cellular integrity and functional responses.
During inflammation, phospholipases, from whatever source, that are normally under the control of natural suppressor systems, are activated to degrade membrane phospholipid which, in turn, generates oxygen free radicals. PLA2 is a key enzyme which is activated in inflammation to metabolize substrate phospholipids and release free fatty acids. These fatty acids (i.e., arachidonate) released by PLA2 are converted to potent biologically active metabolites, lysophospholipids, prostaglandins, and leukotrienes. These are themselves substrates for other enzymes leading to the production of thromboxanes, platelet activating factor and other substances, with the concomitant generation of oxygen free radicals.
Phospholipases, particularly PLA2, as membrane targeted enzymes, play an important role since expression of their activity results in further production of inflammatory mediators leading to membrane injury which propagates damage within the cell itself or to adjacent tissue. Thus, the spread of injury from the initial site to contiguous or distant sites can be promoted by the activation and/or release of PLA2.
In addition to the intrinsic membrane-related tissue breakdown via the activation of PLA2, phospholipases, and particularly PLA2, are part of the normal defense system of the body. PLA2 is found in human white blood cells (WBCs). WBCs play a role in resisting infection, but when these cells are mobilized to ward off injury and infection, PLA2 is released from adherent and circulating WBCs and produces local tissue activation which can increase the extent of initial injury. In addition, WBCs adhere to blood vessel walls where they release enzymes such as PLA2. WBCs also generate free radicals such as superoxide, in large quantities, and thus promote damage to the vascular endothelium, lung alveoli or to tissue sites contiguous with WBC infiltration or concentration. Where inflammation is found, WBCs are usually present in abundance and the WBCs adhere to vascular endothelium, with subsequent release and activation of PLA2 resulting in damage to vascular integrity during shock and ischemia. Thus, in spite of being a prime defense system of the body against infection, WBCs can also damage the body by propagating injury and inflammation.
A classical description of inflammation is redness and swelling with heat and pain. Inflammation has been defined as the reaction of irritated and damaged tissues which still retain vitality. Inflammation is a process which, at one level, can proceed to cell death, tissue necrosis and scarring. At another level, inflammation can be resolved with a return to normalcy and no apparent injury or with minimal changes, i.e., pigmentation, fibrosis or tissue thickening with collagen formation related to healing and scarring.
Microscopically, inflammation has been described as: (1) atony of the muscle coat of the blood vessel wall; (2) endothelial adherence of inflammatory cells followed by migration of these cells from the vascular space into tissue.
The events described above are often mediated by phospholipase activation, followed by fatty acid release and the formation of free radicals. Cytokines, secreted by immune cells, induce PLA2 secretion by their actions on a variety of cells. Interleukin-6 stimulates hepatocytes to increase PLA2 secretion many-fold. Interleukin-1 and tumor necrosis factor induce PLA2 secretion by endothelial cells and by chondrocytes. Thus, immune cell products directly stimulate the hydrolysis of membrane phospholipids and production of arachidonic acid metabolites by a variety of target cells, amplifying the inflammatory response.
Alternatively, increased phospholipase activity can relate to exogenous enzyme released from infecting pathologic organisms such as viruses, bacteria, Rickettsia, protozoa, and fungi. These pathogens often possess phospholipases as factors intrinsic to their infectious activity. In the case of Naegleria fowleri, a pathogenic amoeba with affinity for the brain, destruction of brain membranes induced by phospholipases secreted by Naegleria can occur at sites in the brain distant from where the organism is localized. In another example toxoplasma cannot enter the host cell if its PLA2 enzyme is inhibited by a specific drug. What is needed to treat certain infections, particularly intracellular pathogens, is an effective PLA2 inhibitor. Such an effective PLA2 inhibitor is particularly needed in cases of protozoal infections for which there are few effective antibiotics.
PLA2 is also one of the major toxic components of snake venom. Bites of certain snakes inject venom containing PLA2 into the wound, causing toxic and inflammatory responses which may be lethal. What is needed are inhibitors of PLA2 which may be administered to recipients of snake bites and bites of other animals.
Pathologic effects of phospholipases may be local, regional or systemic. These pathologic effects are governed by the phospholipase enzyme released, the level of albumin, natural inhibitors of enzyme action, and factors of diffusion, circulation and tissue vulnerability based on intrinsic inhibitors or the susceptibility of previously damaged or oxidized membranes or proteins to phospholipase action.
Inflammation is associated with trauma, infection and host defense reactions related to direct bacterial or virus killing by the associated immune responses. In general, immune responses can be both beneficial, protective or tissue damaging as can be seen in their role to promote resistance to infection or cure of infection. Alternatively, immune responses may produce autoimmune phenomena that result in allergy, i.e., asthma, urticaria, in graft versus host disease, in glomerular nephritis, in rheumatic fever, or in lupus and rheumatoid arthritis.
In regard to the current treatment of inflammation, corticosteroids are effective anti-inflammatory agents, but must be used cautiously because they are powerful immunosuppressants and inhibitors of fibroblastic activity necessary for wound healing and bone repair. In addition, corticosteroids have powerful hormonal activities and their toxic side effects involve interference with wound repair and bone matrix formation, sodium retention, potassium loss, bone demineralization, decreased resistance to infection, and diabetes. Corticosteroids also have effects on steroid formation, cataracts, blood pressure, protein utilization, fat distribution, hair growth and body habitus. Alternatively, the clinically active NSAIDs, such as aspirin, indomethacin, ibuprofen, etc., work by inhibiting the conversion of free fatty acids to prostaglandins. The side effects of NSAIDs include gastric ulceration, kidney dysfunction and Reye""s Syndrome. Metabolites of prostaglandin can be either damaging or protective to cells depending on the structure of the prostaglandin produced or utilized pharmacologically and the route of administration, cell or tissue effected.
As discussed previously, in conjunction with fatty acid release, leukotrienes are generated as part of phospholipid cell membrane mediated injury produced by phospholipase activation. These leukotrienes produced from membrane phospholipid breakdown damage tissue through direct toxic action, effects on ionic channels, and associated free radical formation. Leukotrienes also damage tissue by indirect effects on vascular smooth muscle or on the vascular endothelial lining via effects on platelets, WBCs, or endothelial cells, or secondarily through effects on constriction of smooth muscle. Leukotrienes are responsible for smooth muscle constriction leading to bronchospasm and the asthmatic attacks seen in allergy or infectious asthma. Thus, there is an ongoing search for leukotriene inhibitors for clinical application in the treatment of allergy, asthma and tissue injury and inflammation.
Because the phospholipase-activated biochemical pathway for the formation of prostaglandins and leukotrienes derived from free fatty acids is branched, inhibition of one branch of this pathway, as with NSAIDs, can create an imbalance in these reactive metabolites. This imbalance may actually aggravate inflammation and promote cell injury as evidenced by the gastric ulcerative side effects of NSAIDs.
Due to these adverse effects of both steroids and NSAIDs, there is great clinical interest in identifying phospholipase inhibiting agents that do not have steroidal or NSAID side effects, but like corticosteroids modulate the first step leading to the production of injurious metabolites, fatty acids and free radicals.
Free radicals, produced by white blood cells, tissue injury or metabolic processes, are highly reactive chemical species which, in the case of tissue injury, are most often derived from respiratory oxygen. Oxygen, while necessary for energetics of life, is also a toxin which, as the chemically related superoxide, or as peroxides, can damage tissue instead of supporting it. Free radicals derived from oxygen are critical to damage produced by radiation, inflammation, reperfusion tissue injury or through excess oxygen inhalation or exposure. Free radicals are used by white blood cells to destroy infecting organisms, but can, under circumstances of shock, infection and ischemia, damage or destroy the tissue they were meant to protect. Free radicals, induced by radiation, oxygen exposure, chemical agents (i.e., alkylating agents, dioxin, paraquat) or white blood cell reactions may damage tissue or be involved in mutational changes associated with aging, radiation or chemotherapy injury, the development of cancer, and hyperimmune proliferative disease such as rheumatoid arthritis. In addition, these reactive chemical species can, through oxidation of proteins, enhance the vulnerability of proteins to protease digestion.
The exact pathologic mechanisms of many skin inflammations, such as atopic dermatitis, are not clear, but probably involve inflammatory cells which can secrete or respond to PLA2. Allergic diseases involve tissue mast cells, which can be primed or triggered by PLA2 for the release of their inflammatory granule contents, such as histamine. These cells also release additional PLA2. What is needed are inhibitors of PLA2 that adequately penetrate skin after topical application and possess prolonged anti-PLA2 activity.
Previous published studies have demonstrated high levels of a proinflammatory PLA2 in human herniated vertebral discs. The isolated enzyme is toxic to dorsal root ganglion cells in culture and excised sciatic nerve. While not wanting to be bound by this statement, it is believed that PLA2 may mediate inflammation and nerve tissue damage in spinal cord injury and in sciatic nerve inflammation and may also mediate a variety of neurological inflammatory conditions. Recently, Stephenson et al., (Neurobiology of Disease 3:51-63 (1996) have observed elevated cytosolic PLA2 activity in brains with Alzheimer""s disease.
PLA2 also has the capability to induce severe, delayed neurotoxicity syndrome, including extensive cortical and subcortical injury to forebrain neurons and fiber pathways, when injected intracerebroventricularly as described by Clapp et al. (Brain Research 693:101-111,1995) the entirety of which is incorporated herein by reference. We have also observed that preparations of PLA2 and homogenates of human vertebral disks containing extracts of the nucleus pulposus are inflammatory when injected into the mouse paw and induce edema. Edema induced by human disk homogenate is maximal between 1-3 hrs and remains so for at least 6 hrs. These results support the hypothesis that leakage of nucleus pulposus from a herniated disk may promote inflammation in human disk disease. Accordingly, what is needed are inhibitors of PLA2 mediated inflammatory processes. Such inhibitors should alleviate the inflammation and resultant pain and discomfort associated with disk disease and other neurological inflammatory conditions.
Tissues that are excised from animals for subsequent transplantation into recipients often display damage following transplantation during reperfusion of ischemic tissue. Both ischemia and reperfusion increase PLA2 activity and release leading to inflammatory processes with marked activation of the vascular endothelium. These processes decrease the probability of successful transplantation thereby increasing the incidence of rejection and the need for additional immunosuppressive therapy. Such problems greatly increase morbidity and mortality, increase the costs of treatment and insurance, and result in lost time at work. What is needed are drugs that will inhibit PLA2 activity and enhance tissue preservation before transplantation thereby decreasing ischemia reperfusion injury.
Infections caused by parasites constitute a major public health problem throughout the world for humans and animals, annually resulting in significant incidence of disease, suffering and death. Parasites such as those that cause malaria and other protozoal parasites of animals and humans are especially troublesome. We have found that the PLA2 inhibitor, quinacrine (mepacrine), significantly reduced molting of larval forms of an animal filarid. What is needed are new compounds that effectively inhibit PLA2 activity for application to parasites such as those causing malaria and other protozoal parasites injurious to animals and humans.
A previous study by Clay et al. (Third International Congress: Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Radiation Repair, Abstract #162) reported that the product of PLA2 activation, 1-acyl lysophospholipid, which affects membrane fluidity, accumulates in stored blood and may be taken up by white blood cells (WBCs) and used to make platelet activating factor (PAF) thereby xe2x80x9cprimingxe2x80x9d WBCs during storage and promoting injury during subsequent transfusion. It has been suggested that increased PLA2 activity may perturb cells in storage. What is needed are compounds that protect blood cells and other cells during storage so that these cells will not cause problems when utilized.
Accordingly, what is needed are compounds and methods of using these compounds which provide protection against the deleterious effects of PLA2 activation. These compounds should be capable of inhibiting PLA2, thereby decreasing the PLA2 metabolites which are substrates for the cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, and other enzymatic pathways which lead to formation of cyclic endooperoxides, prostaglandins (such as prostacyclin and thromboxane), leukotrienes, and platelet activating factor. These compounds should decrease inflammatory processes and free radical production in a variety of tissues and cells. They should be capable of being administered in vivo (topically, orally, by injection and through other means), ex vivo and in vitro and should also exhibit low or no toxicity. These compounds should display different solubilities in lipid and aqueous systems depending on the mode of application and the desired target.
The present invention provides both lipid and/or water soluble compounds that are PLA2 inhibitors having antioxidant properties and/or antiinflammatory properties. This invention provides bioactive compounds which are oligomers (dimers, trimers and tetramers, etc.) of fatty moieties that inhibit PLA2 activity. The terms dimer, trimer, tetramer and pentamer as used throughout the present description define the number of fatty moieties present in the particular molecule. That is to say a dimer has two fatty moieties, a trimer has three, a tetramer has four, a pentamer has five, etc. The compounds of the present invention possess at least one double bond to enhance their anti-inflammatory and cytoprotective or tissue protective effects.
The compounds of the present invention may be used for treating or prophylactically inhibiting phospholipase mediated injury and/or injury due to oxidation. In a specific sense, the present invention provides agents for preventing and/or treating inflammation and cell destruction in mammalian tissue and for protection and preservation of biologic material such as cells, tissues, organs and fluids obtained from animals and humans. The present invention also provides agents for protection and preservation of food obtained from animals and plants, and for cellulose products and wood products obtained from plants. The compounds of the present invention protect phospholipid cell membranes, and proteins from the effects of oxidative injury or aging. These compounds of the present invention also inhibit free radical reactions and thereby stabilize proteins for maintenance of biologic half-life, anti-coagulant activity, and food preservation.
More specifically, the present invention provides pharmacologically active, anti-phospholipase compounds. These compounds are soluble and/or dispersible in a suitable carrier. The compounds may exist as oligomers such as dimers, trimers, tetramers etc., or as combinations thereof. In accordance with the present invention, the compounds have at least two fatty moieties and contain at least one unsaturated double bond. Each fatty moiety may take a variety of forms, may possess the same or different functional groups, may be a different length. In one preferred form, the compounds of the present invention may have an acid group or any salt form thereof. The compounds of the present invention may also be present in ionized form.
Accordingly, an object of the present invention is to provide compositions that inhibit the activity of the enzyme PLA2.
It is a related object of the ,present invention to decrease levels of products of the enzymes cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, prostacyclin oxycyclase, thromboxane synthase, and prostaglandin isomerase pathways by inhibiting the activity of PLA2.
Another object of the present invention is to provide methods of synthesizing these compositions that inhibit the activity of PLA2.
It is also an object of the present invention to provide methods of treating conditions associated with PLA2 activity.
Yet another object of the present invention is to provide a composition and treatment for oxidative and free radical damage to cells, tissues and organs in vitro and in vivo.
Another object of the present invention is to provide methods of applying an effective amount of the compositions to treat inflammation.
Another object of the present invention is to provide a composition and treatment for inflammatory processes.
It is also an object of the present invention to provide oral and topical treatments for arthritis with the compositions of the present invention.
Yet another object of the present invention is to provide treatments for pain using the composition of the present invention.
Another object of the present invention is to provide oral and topical treatments comprising administration of an effective amount of the compositions of the present invention, for a variety of conditions, such conditions including, but not limited to, the following: reflex sympathetic dystrophy; inflammation of the central and peripheral nervous system, diseases related to inflammation of the nervous system including, but not limited to, Alzheimer""s disease, inflammation of spinal nerves, autonomic nerves and cranial nerves; inflammatory radiculopathy; back pain including low back pain; myo-fascial pain syndromes; inflammation of the connective tissues, including meninges, inflamed and diseased facet joints, herniated disks, diseased disks, torn and injured annulus fibrosis, diseases of the joints, ligaments, cartilage and synovial membranes; mastocytosis; shock including septic shock, anaphylactic shock, anaphylactic shock resulting from radiocontrast administration, and shock resulting from bacterial infections; bacterial infections; uremia; autoimmune disorders; parasitic infections including, but not limited to, malaria; inflammation including allergic inflammation; skin inflammation, itching, and other dermatologic disorders due to allergic reactions, dry skin, erythema, solar, nuclear and other forms of radiation, windburn, acne, psoriasis, eczema, reactions to chemicals and toxins, contact dermatitis, and reactions to plants including, but not limited to, poison ivy, poison oak, poison sumac; bites of insects including, but not limited to, mosquitos, fire ants, chiggers, ticks, bees, spiders, fleas and flies; bites of reptiles, especially venomous reptiles, amphibians, and other animals; contact with various animals with venom on their skin such as poisonous frogs; pruritis associated with local dermatologic or systemic disease; prevention of tissue ischemia including tissue in vivo and tissue destined for transplantation, prevention of ischemia-reperfusion injury, prevention of ischemia-reperfusion injury in the setting of resuscitation from hypovolemic shock, renal ischemia, myocardial infarction, angina, and cardiac ischemia; endothelial inflammation, cardiotoxicity associated with administration of anti-cancer compositions, inhibition of coronary or cerebral restenosis following angioplastic or other vascular procedures, inhibition of platelet activity, especially in vessels following various procedures such as angioplasty and after insertion of catheters, shunts and other devices, inhibition of thrombin-activated platelet aggregation; pulmonary diseases including, but not limited to, asthma, cystic fibrosis, inflammation of the lungs secondary to ischemia of the gastrointestinal system, adult respiratory distress syndrome, and other allergic and inflammatory reactions of the pulmonary system including inflammation of the tissues of the upper respiratory system, allergic rhinitis, and respiratory distress syndrome; inflammation of the gastrointestinal system including, but not limited to, Crohn""s disease, eosinophilic gastroenteritis, peritonitis, ulcerative colitis, ulcers of the small bowel and stomach, esophagitis, inflammation of the stomach, inflammatory bowel disease; ocular inflammation; preservation of whole blood; preservation of tissues, cells, and organs for transplantation; and protection of mitochondria.
Another object of the present invention is to provide a method of applying an effective amount of the compositions of the present invention through injection, topical, oral, or aerosol administration, for the treatment of inflammation resulting from the bites of insects, reptiles, amphibians, and other animals, especially venomous animals, such as venomous snakes.
Another object of the present invention is to provide a composition and method for inhibition of platelet function.
It is an object of the present invention to provide a composition for prevention and treatment for acute and chronic rejection of transplants, and for treatment of graft-vs-host disease and autoimmune diseases.
It is another object of the present invention to provide a composition for the treatment of neoplastic disease.
It is another object of the present invention to provide an easy to use topical therapeutic composition and topical treatment for various forms of arthritis and other inflammatory diseases, including, but not limited to, rheumatoid arthritis, inflammatory arthropathies, osteoarthritis, gout, and lupus.
It is another object of the present invention to provide a composition and treatment for parasitic infections including, but not limited to, toxoplasmosis, malaria, Naegleria fowleri, Dilofilaria immitis, nematodes, and pathogenic protozoans such as toxoplasma gondii, falciparum malaria, amebiasis, amoeba, and cryptosporidia.
It is another object of the present invention to provide the enhanced range of motion and reduced pain provided to patients with reflex sympathetic dystrophy following topical or oral application of the compositions of the present invention.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description.
The following applications are hereby incorporated by reference in their entirety: application Ser. No. 08/467,690 filed Jun. 6, 1995; application Ser. No. 08/475,335 filed Jun. 7, 1995; application Ser. No. 08/010,456 filed Jan. 27, 1993; application Ser. No. 07/839,780 filed Apr. 1, 1992; application Ser. No. PCT/US90/04615 filed Aug. 16, 1990; application Ser. No. 07/399,941 filed Aug. 29, 1989; application Ser. No. 07/256,330 filed Oct. 11, 1988; application Ser. No. 07/156,739 filed Feb. 18, 1988; and application Ser. No. PCT/US87/00408 filed Feb. 24, 1987.
Cis-unsaturated, but not saturated fatty acids, inhibit in vitro PLA2 activities derived from human platelets and human polymorphonuclear leukocytes (PMNs). PLA2 activity is inhibited by oleic, linoleic, and arachidonic acids to approximately the same extent indicating that the presence of a single cis-double bond is as inhibitory as multiple cis-double bonds. In contrast, fatty acids containing trans-double bonds or methyl esters of cis-unsaturated fatty acids are less inhibitory of PLA2 activity. Thus, it is hypothesized that the preferred structural characteristics for inhibition of in vitro PLA2 activity by unesterified fatty acids include at least one double bond. Oleic acid inhibits in vitro PLA2 activity due to the presence of a single double bond at the C-9 position. Oxidation of the sarcoplasmic reticulum of muscle and of phospholipid membranes predisposes them to phospholipase degradation. Phospholipid membranes that have been oxidized at particular sites may appear intact and maintain functional activity, but their oxidation makes them vulnerable to degradation and destruction by PLA2 or other phospholipases from endogenous or exogenous sources.
The observations illustrating the enhanced vulnerability of phospholipid membranes to phospholipase following oxidative and free radical mediated changes in cell membranes and/or cisunsaturated fatty acids have been employed in accordance with the present invention in the design of novel anti-inflammatory and cytoprotective agents. The present invention thus provides a biochemical and synthetic organic approach to controlling the expression of PLA2 enzymes which is vital to the maintenance of membrane structure.
Although not wanting to be bound by the following hypothesis, it is believed that the number of available methylene interrupted unsaturated double bonds is directly related to the susceptibility of fatty acids to oxidation. This governs the ability of unsaturated fatty acids to act as anti-oxidants. This property, in conjunction with the anti-PLA2 activity of the fatty moiety compounds of the present invention, markedly expands the scope of the anti-inflammatory and cytoprotective activity of the new agents disclosed herein. It is the property of the dual action of those compounds, i.e., their action as PLA2 inhibitors with varying anti-oxidant activity, that provides the spectrum of anti-inflammatory activity in model systems that have direct applicability to cytoprotection and the control of inflammation and pathophysiology.
A nonfunctional alkyl chain or group is known as a hydrocarbon group because it contains only Cxe2x80x94C and Cxe2x80x94H single bonds. A nonfunctional alkyl chain therefore contains the maximum possible number of hydrogens per carbon which may be represented as xe2x80x94CnH2n+1. A functionalized alkyl chain or group has substituted for one or more hydrogens on the alkyl chain, one or more atoms or groups of atoms that have characteristic chemical behavior. These atoms or groups of atoms that have characteristic chemical behavior are also known as functional groups. Included in these functional groups are Cxe2x95x90C, OH, COOH, SO3H, PO3H, NH2, xe2x80x94Oxe2x80x94, and halides,
In summary, a single double bond in a fatty moiety compound is sufficient to inhibit PLA2 activity in vitro and in situ. The addition of multiple double bonds provides the additional value of an increase in potent anti-oxidant activity along with PLA2 inhibitory action. The present invention thus provides compounds characterized by both anti-PLA2 and varying anti-oxidant activity to maximize the anti-inflammatory and cytoprotective action which is the key to the clinical value of the compounds of the present invention.
In addition to inhibiting PLA2 activity, the anti-oxidant action of these compounds protects proteins that become increasingly vunerable to attack by proteases due to oxidation. Thus, the cytoprotective PLA2 inhibitors of the present invention, which have anti-oxidant activity as well, have value both in stabilizing membrane phospholipid and in inhibiting or preventing protein degradation or denaturation. This suggests that the compounds of the present invention act to minimize inflammation at its onset and will also interrupt the inflammatory process in progress.
The compounds of the present invention block arachidonic acid release from human polymorphonuclear cells and endothelial cells, a reaction mediated by cellular and secretory PLA2 activity. Thus, the compounds of the present invention are potent, reversible PLA2 inhibitors, and, as such, these agents inhibit the proinflammatory response of the human PMN and other inflammatory cells by inhibition of cellular and secretory PLA2 activity. In addition, the compounds of the present invention inhibit, to various extents, the free radical activity in cells and tissues involved in the inflammatory process.
In a general sense, the present invention provides pharmacologically active, anti-phospholipase compounds. Preferably the compounds of the present invention are water or lipid soluble antioxidants. The preferred compounds have at least two fatty moieties and contain at least one unsaturated double bond. The fatty moieties may be different from each other in several features including, but not limited to, chemical composition, functional groups, the degree of unsaturation, and the length of the hydrocarbon chain. The compounds may also have at least one organic group, one active acid group or any salt form or ionized form thereof. The invention contemplates a variety of configurations including oligomers such as dimers, trimers, tetramers, and combinations thereof. Several of these compounds provided in accordance with principles and concepts of the present invention may be prepared as outlined in the following specific examples.
The compounds of the present invention are not generally hydrolyzed by pancreatic enzymes and are different from glycerol-based compounds in terms of their chemistry and metabolism. For example, we have observed that two compounds of the present invention; PX-13 and PX-18, are resistant to degradation in vitro by the commercially available pancreatic enzyme preparation, pancreatin, obtained from Sigma Chemical Company (St. Louis, Mo.). The resistance of PX-13 and PX-18 to metabolism by pancreatic enzymes supports their stability after oral administration.
The oligomers (dimers, trimers, tetramers, pentamers, etc.) of unsaturated fatty acids and the other compounds having at least one unsaturated straight chain fatty radical as described above, affect fundamental membrane phospholipid reactions of phospholipase-induced degradation and free radical peroxidation. The data set forth herein confirm with experimental results that these compounds are potent anti-inflammatory and cytoprotective agents.
The appropriate dosage of an effective amount of the unsaturated fatty moiety compounds of the present invention for treatment of mammals including humans, against phospholipase mediated injury and/or inflammation should be in the range of approximately 1 to 75 mg per kg (mg/kg) of body weight and preferably approximately 2 to 50 mg/kg of body weight, with a more preferable range of approximately 10 to 40 mg/kg of body weight, when the compound is administered orally or intraperitoneally (IP). When administered intravenously the dosage should be approximately 50% of the oral or IP dosage to achieve the same level of the drug in the blood. The described dosage is also be appropriate for prevention of human platelet aggregation or blood clotting. As is known to those skilled in the art, therapeutic doses expressed in terms of amounts per kilogram of body weight or surface area may be extrapolated from mammal to mammal, including to human beings. The compounds of the present invention may also be administered in an aerosol manner, such as an intranasal spray to treat inflammation of the nasal cavities, nasopharynx and adjacent regions or as inhaled formulations to treat inflammation of the upper and lower respiratory system.
The compositions of the present invention may be formulated for administration in any convenient way by analogy with other topical compositions adapted for use in mammals. These compositions may be used in any conventional manner with the aid of any of a wide variety of pharmaceutical carriers or vehicles. The compounds of the present invention described above can be provided as pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal (including ionophoretic administration), buccal, oral, rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the combinations may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor. The biodegradable polymers and their use are described, for example, in detail in Brem et al., J. Neurosurg. 74:441-446 (1991).
A pharmaceutically acceptable solvent is one which is substantially non-toxic and non-irritating under the conditions used and may be readily formulated into any of the classical drug formulations such as powders, creams, ointments, lotions, gels, foams, aerosols, solutions and the like. Particularly suitable solvents include, but are not limited to, water, ethanol, acetone, glycerin, propylene carbonate, dimethylsulfoxide (DMSO), and glycols such as 1,2-propylene diol, i.e., propylene glycol, 1,3-propylene diol, polyethylene glycol having a molecular weight of from 100 to 10,000, dipropylene glycol, etc. and mixtures of the aforementioned solvents with each other.
A topical cream may be prepared as a semi-solid emulsion of oil in water or water in oil. A cream base formulation by definition is an emulsion, which is a two-phase system with one liquid (for example fats or oils) being dispersed as small globules in another substance (e.g., a glycol-water solvent phase) which may be employed as the primary solvent. The cream formulation may contain fatty alcohols, surfactants, mineral oil or petrolatum and other typical pharmaceutical adjuvants such as anti-oxidants, antiseptics, or compatible adjuvants. A typical cream base formulation is as follows:
A xe2x80x9cclassicalxe2x80x9d ointment is a semi-solid anhydrous composition which may contain mineral oil, white petrolatum, a suitable solvent such as a glycol and may include propylene carbonate and other pharmaceutically suitable additives such as surfactants, for example Span and Tween, or wool fat (lanolin), along with stabilizers such as antioxidants and other adjuvants as mentioned before. Following is an example of a typical xe2x80x9cclassicalxe2x80x9d ointment base:
Additionally, the compounds may be formulated as oral, parenteral, subcutaneous, intravenous, intraarticular, intramuscular, intraperitoneal, intralesional and otherwise systemic compositions. Depending on the intended mode, the compositions may be in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for single administration of precise dosages.
The compositions will include a conventional pharmaceutical carrier or excipient and a therapeutically effective amount of a compound of the present invention and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
The amount of active compound administered will, of course, be dependent on the human or animal subject being treated, the severity of the affliction, the manner of administration and the judgment of the prescribing clinician.
Typical compositions contain approximately 0.01-95% by weight of active ingredient, with the balance one or more acceptable non-toxic carriers. The percentage of active ingredient, will, of course, depend upon the dosage form and the mode of administration.
For solid compositions, conventional non-toxic solid carriers including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols and propylene glycol, as the carrier. Liquid pharmaceutically administerable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine, sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art, for example, see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.
For oral administration of the compounds of the present invention, a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain 2%-95% active ingredient, preferably 5%-90%.
Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
Ointments for topical application may be prepared by incorporating approximately 0.1 to 10% of the compound as an oil or salt form into an ointment base containing emulsifying agents such as stearic acid, triethanolamine and/or cetyl alcohol. The formulation may also include ingredients such as glycerol, petrolatum, water and preservatives as required.
Water based lotions may contain the compounds of the present invention as an oil or solid in amounts ranging from approximately 0.1% to 5.0% by volume. Such lotions may contain glycerine and/or bentonite as suspending agents as is well known in the art. The present invention may also be incorporated into creams.
The compounds may also be incorporated into classical (one or two phase) or non-classical (aqueous emulsion) aerosol formulations. Such formulations include the compounds and an appropriate propellant carrier in which the compounds are dissolved or dispensed. In the classical form the active ingredients are generally used as an oil dispersion or in solution in an organic solvent such as ethanol. In the non-classical form the active ingredient is dissolved in water. In each case the concentration of the active ingredient in the carrier may be about approximately 0.1 to 10% by weight or volume.
Of particular advantage is the fact that the unsaturated straight chain fatty moiety compounds described above function pharmacologically at the site of inhibitory action for the arachidonate cascade and preferentially affect stimulus-induced mobilization of arachidonate. Inhibition of PLA2 depresses the production of both prostaglandins and leukotrienes in stimulated or inflamed cells. Importantly, the compounds described above have a much more pronounced effect on stimulus-induced, than on controlled release of arachidonate indicating a selective effect on the former. Moreover, when phospholipids are peroxidized, the polymer compounds described above are capable of inhibiting the degradation of such lipids by lysosomal phospholipase C, indicating that these compounds can protect already damaged (oxidized) membranes.
Thus multiple actions are responsible for the anti-inflammatory activity of the fatty moiety compounds of the present invention, and on the basis of inflammatory models, it is evident that these compounds can effectively rival or replace both currently available steroids and NSAIDs in the treatment of inflammation, making the fatty moiety compounds of the present invention candidates for clinical application and usefulness in localized and systemic injury and disease.
The fatty moiety compounds described above, by protecting lipid membranes and possessing anti-oxidant activity, are potent anti-oxidants for preservation, not only of living cells and tissues, but their action makes them effective as preservatives of biological materials from animal, human or plant origin, and as preservatives of chemical agents subject to oxidative injury. For purposes of protecting and preserving biological materials subject to oxidation injury, the fatty moiety compounds of the present invention may be used at concentrations of approximately 0.1 to 100 xcexcM. These molarities are calculated as the molarity that would be obtained if the drug were dissolved in a weight of water which is the same as the weight of the biological material to be preserved. For example, in vitro, anti-oxidant and/or anti-phospholipase applications, concentrations of from about approximately 0.1 to about 500 xcexcM should be effective.
This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.